Polyhydroxy butyrate biosynthesis by Azotobacter chroococcum MTCC 3858 through groundnut shell as lignocellulosic feedstock using resource surface methodology

This work appraises the prospect of utilising groundnut shell hydrolysate as a feedstock used for PHB biosynthesis by Azotobacter chroococcum MTCC 3853 under SMF conditions. Sugar reduction: untreated and pretreated 20% H2SO4 (39.46 g/l and 62.96 g/l, respectively), untreated and enzymatic hydrolysis (142.35 mg/g and 568.94 mg/g). The RSM-CCD optimization method was used to generate augment PHB biosynthesis from groundnut shell hydrolysate (30 g/l), ammonium sulphate (1.5 g/l), ammonium chloride (1.5 g/l), peptone (1.5 g/l), pH 7, 30 °C, and a 48 h incubation time. The most convincing factors (p < 0.0001), coefficient R2 values of biomass 0.9110 and PHB yield 0.9261, PHB production, highest biomass (17.23 g/l), PHB Yield(11.46 g/l), and 66.51 (wt% DCW) values were recorded. The control (untreated GN) PHB yield value of 2.86 g/l increased up to fourfold in pretreated GN. TGA results in a melting range in the peak perceived at 270.55 °C and a DSC peak range of 172.17 °C, correspondingly. According to the results, it furnishes an efficient agricultural waste executive approach by diminishing the production expenditure. It reinforces the production of PHB, thereby shrinking our reliance on fossil fuel-based plastics.

RSM-CCD optimization process. The optimization results for increasing PHB production are shown in Table 3. RSM CCD design in each independent variable was coded at three levels of low, middle, and high, and the four factors. The model data given to the variables are coded values and the actual value levels. The CCD design ran thirty experiments, and regression on the empirical data was successful. The equations of polynomials were analysed and converted into empirical and presumed values.
The ANOVA results noted the value of the probability model F > 0.0001 and revealed that the quadratic model was the most significant. The F-values for biomass of 10.96 (Table 4), and PHB Yield of 13.43 (Table 5), suggest the model is significant. The highest coefficient of determination was revealed, with R2 values of biomass of 0.9110 and a PHB yield of 0.9261. The predicted R squared values for biomass were 0.2901, 0.3954 for PHB Yield, while the adjusted R-squared values were 0.8279, 0.8571 for PHB Yield. Adequate precision response results when the ratio of greater than 4 is desirable. The ratio of biomass yield is 16.0908, the PHB yield is 16.4732, which indicates an adequate signal and the coefficient value of > 10 is tolerable. The correlation between empirical and presumed values was good. The model was validated successfully.
In the present work, the regression equation was observed to have the maximum PHB production. GN hydrolysate (30 g/l), ammonium sulphate (1.5 g/l), ammonium chloride (1.5 g/l), peptone (1.5 g/l), pH 7, temperature 30 °C, incubation time 48 h, biomass yield (CDW) 17.23 ± 0.12 g/l, PHB Yield 11.46 ± 0.5 g/l and 66.51% PHB accumulation, with 95% conformity with the assumption were the factors. Untreated GN biomass yield  (Table S2). The PHB production and optimization processes were repeated three times. The model was validated and optimum results for maximum PHB production and their interactions, contour plots, and RSM 3D curves were plotted in Fig. 1.

Characterization of PHB by A. chroococcum. FT-IR analysis.
In this present work, purified PHB was analyzed for functional groups through FTIR spectroscopy results. The peak value is noted in Fig. 2 NMR analysis. In this present work, the production and purified form of PHB from GNhydrolysate residues were analyzed by using 1 H NMR spectroscopy graph plotted in Fig. 3a. The NMR spectrum (400 MHz, CDCl3) value of PHB agreed with various carbon atoms. It clarified with dominant peaks at 1.248 ppm attained from the absorption of the CH3 group; another peak at 2.502 ppm from the absorption of the CH2 group, and a 5.228 ppm peak for methane (CH) groups. 13 C NMR spectrum analysis (400 MHz, CDCl3) yielded peak results of 19.74 ppm due to CH3 (methyl group), 40.87 ppm due to CH2 (methylene), 67.45 ppm due to CH (methane), and 169.19 ppm due to C=O (carbonyl) functional group (Fig. 3b).   (Fig. 4b).

Discussion
Recently, researchers have investigated the PHB of biosynthesis from lignocellulose residues as a substrate under laboratory conditions 3 . This study, which produced PHB strain A. chroococcum in a pre-screened process, was carried out by the Sudan Black method 24 . In the acid hydrolysis process, 15% of the peanut shell was added   27 . Wheat bran mixed with β-glucosidase of Aspergillus niger (50 CBU/g) to reduce the sugar yield of untreated and pretreated wheat bran (159 and 629.1 mg/g) respectively 28 . Enzymatic pretreatment of the yield of reducing sugars to obtained in 72.67 mg/g of untreated rice husk and 266.5 mg/g of pretreated rice husk 29 .
In present study RSM CCD was optimized to biosynthesis of PHB fourfold increased, highest biomass (17.23 g/l), PHB Yield (11.46 g/l), 66.51 (wt% DCW) and R2 values biomass 0.9110 and PHB yield 0.9261 were recorded. Similarly production of PHB from peanut shell by Bacillus sp to produced highest PHB (945-1205 mg −1 ) and Yield (55-65% w⁄w) 29 . Coconut coir as an agricultural substrate produced PHB content at 64.7% (w/w) and an improved 3.7-fold in PHB yields 16 . Maximum production of PHA from cardboard industry waste water by Enterococcus sp. NAP11 produced 79.27% and Brevundimonas sp. NAC1 produced 77.63% and PHA concentration of 5.236 g/l and 4.042 g/l 30 . Produced biosynthesis of PHB from pulp waste, biosynthesizing 61.7% of DCW reported that R2 values (0.9837 and 0.9735) 31 . Similar results RSM Optimization process to improve the production of PHB from rice bran, PHB yield attained the value of F = 14.389 and p < 0.005 for the strong significant model and R 2 = 0.963 32 . PHB biosynthesised from banana peel extract by marine seaweeds   33 .
In the TGA section of this study, the PHB from the selected candidate strain thermal degradation peak value was 270.55 °C and completely decomposed at 337.98 °C. Previous studies have discovered that the PHB TGA result, the PHB degrade temperature of 288 °C, the ester bond breaks, and the PHB sample completely degrades at 320°C 36 . Previous research has shown that these results are comparable to PHB-synthesized endothermic peak values at 178°C 17 . TGA analysis of the peak melting temperature of PHB at 260 °C was obtained 41 . In the DSC section of this study, the PHB from the chosen candidate strain had a melting temperature range of 172.17 °C and a Tg of 5.2. Similarly, the extracted PHB in DSC analysis of the melting temperature (Tm) was 171.8 and the glass transition temperature (Tg) was 4.03 °C, respectively 36 . DSC analysis extracted the PHB melting point peak value at 171 °C 41 . Another study found similar results in DSC analysis of the melting temperature range and PHB Tm peak at 175 °C (water hyacinth-hexose rich (or enzymatic) hydrolysate), 173 °C (water hyacinth-pentose rich (or acid) hydrolysate), 177 °C (P. hysterophorus-hexose rich (or enzymatic) hydrolysate), 173 °C (P. hysterophorus-pentose rich (or acid) hydrolysate) 42 .

Conclusion
In this present study, it could be assumed that enhanced production of PHB biosynthesis from cheap, easily available groundnut shell hydrolysate lignocellulosic residues. PHB increased fourfold in 30 g/l of GN hydrolysate by A. chroococcum produces the maximum results biomass 17.23 ± 0.5 g/l, PHB Yield 11.46 ± 0.02 g/l and 66.51% of PHB. It confirmed that the characterization of PHB was carried out by methods such as FT-IR, NMR, TGA, and DSC and that various parameters were used for fermentation and RSM optimization. It was decided to control environmental pollution and health hazards while decreasing the non-degradable manufacture of polymers. RSM design Groundnut shell hydrolysate successfully well-established as an encouraging statistical tool to enhance PHB yield from GNhydrolysate by A. chroococcum MTCC 3853, which can be utilized to pilot scale up the production of PHB bio-polymers of industrial interest.

Materials and methods
The Microbial Type Culture Collection, Chandigarh, India, purchased the strain A.chroococcum MTCC 3853 used for the fermentation study. Cultivated it in nutrient broth by incubating it at 30 °C and storing the culture in glycerol (1:1, v: v) at − 40 °C.

Pretreatment of GN.
The lignocellulosic raw material GN hydrolysate was collected from the regional agricultural field of Kovilpatti, Tamilnadu, India. GN are washed in tap water several times to remove impurities like sand and dried in an oven at 60 °C, followed by grinding it properly to a fine powder (a thickness of 0.5 mm) and storing it in sterile containers at room temperature for future experiments.
Acid and enzymatic hydrolysis of GN hydrolysate.  Extraction and quantification of polyhydroxybutyrate from GNhydrolysate. Harvested PHB cells were extracted and quantified in accordance with the slightly modified methodology using NaOCl and H 2 SO 4 46,48 . After centrifugation, purified PHB was collected, and this procedure was performed in triplicates at 4 °C to store the sample for further studies.
Characterization of biosynthesized PHB from GNhydrolysate. Fourier transform-infrared spectroscopy (FTIR) analysis. The extracted PHB functional groups were decisive through FTIR spectroscopic analysis as per the methodology 34-36 . 2 mg of extracted PHB sample was mixed with 200 mg of potassium bromide (KBr) and the spectrum peak was estimated at 500 to 4000 cm −1 using the Shimadzu model (RF6000).
Nuclear magnetic resonance spectroscopic analysis. The characterization of PHB was carried out through 1H & 13C NMR analysis [38][39][40] . The 1H & 13 C NMR spectrum analysis was seized at 400 MHz using the Bruker Advance Model. The PHB sample was dissolved in 10 mg/ml −1 of deuteron chloroform (CDCl3) solvent used to interpret it.
Thermogravimetric and differential scanning calorimetry of biosynthesized PHB from GN hydrolysis. The thermal stability of the extracted PHB was further characterised through TG and DSC analysis 36,41,42 . 5 mg of sample and a temperature range of 30-500 °C, with a boiling rate of 10 °C/min in an N gas atmosphere (nitrogen flow rate of 40 ml/min). TG and DSC were determined by Netzsch, model STA 449F3. Melting temperature (Tm), glass transition (Tg), crystallization (Tc) were determined. X c = ΔHf/ω. H f o × 100 where heat of fusion of the samples (ΔHf), the heat of fusion (H f o ) for 100% crystallized PHB (146 J/g) and the mass fraction (ω) for the PHB biopolymer.